The objective of the proposed research is to elucidate the steps which lead mesenchyme cells to differentiate into cartilage tissue. Previous studies suggest an hypothetical sequence involving 1) the early organization of cells into specific skeletal primordia, 2) interaction between cells in the primordia, leading to increased levels of intracellular cAMP, and 3) the onset of chondrogenesis in response to changes in cAMP levels. The proposed studies aim to test further and develop in more detail this hypothetical sequence. Morphological studies, utilizing scanning and transmission electron microscopy, will be carried out in the formation of specific skeletal primordia in normal and several mutant mice. The mutants (short ear, droopy ear and brachypod) all have defects in early stages in the formation of skeletal primordia, but have histologically normal cartilage. Mesenchyme from these mutants will also be used experimentally in tissue cultures to help identify distinct steps in cartilage differentiation. Experiments on chimeric cultures of chick, mouse or quail mesenchyme from different limb regions or mutants will be performed to identify developmentally distinct sub-populations of cells and distinct steps in cartilage tissue formation. Cartilage will be recognized by specific staining methods and immunofluorescence with antibodies to specific collagen types. Mesenchyme from various sources will cultured under different conditions which lead to cartilage development, including treatment with dibutyryl cyclic AMP, in order to study the mechanism of cell interaction and its effects on mesenchyme which lead to chondrogenesis. Also, the effects of these treatments on cyclic AMP dependent protein kinases and their phosphoprotein substrates will be studied in order to obtain information on intracellular events which lead to chondrogenic expression.